Whole-mount In Situ Hybridization Research Articles

Characterization and expression pattern of ZB and PS transposons in zebrafish

Despite comprising much of the genome, transposons were once thought of as junk. However, transposons play many roles in the eukaryotic genome, such as providing new proteins as domesticated genes, expressing during germline-soma differentiation, function in DNA rearrangement in the offspring, and so on. We sought to describe the distribution and structural organization of the two autonomous transposons (ZB and PS) in the zebrafish genome and examine their expression patterns in embryos and adult tissues. The intact copy of ZB and PS was queried by BLAST on NCBI and ENSEMBL using default parameters. Of the copies with coverage and identity, more than 90 % were downloaded to do structural analysis. Spatial and temporal expression patterns were detected by qRT-PCR and Whole-mount in situ hybridization (WISH). There are 19 intact copies of ZB, encoding 341 amino acid residues with DD34E catalytic domain and flanked by 201bp TIRs, and seven intact PS copies, containing 425 amino acid residues with DD35D catalytic domain flanked by 28bp TIRs, were detected in the genome of zebrafish respectively. Analysis of genomic insertions indicated that both ZB and PS transposons are prone to be retained in the intron and intergenic regions of the zebrafish genome. The sense and antisense transcripts of ZB and PS were detected during embryonic development stages and exhibited similar expression patterns. The difference is that the sense strand transcript of ZB was explicitly expressed in midbrain-hindbrain boundary (MHB) and otic vesicle (OV), and pharyngeal arches and pharyngeal pouches (PA&PP) at 48 hpf. In adult zebrafish, the expressions of ZB and PS in muscle and brain are much higher than in other tissues. Our study results indicate that ZB and PS transposons may be involved in the embryonic development and regulation of somatic cells of certain adult tissues, such as the brain and muscle.

The Threshold Effects of Low-Dose-Rate Radiation on miRNA-Mediated Neurodevelopment of Zebrafish.

The biological effects and regulatory mechanisms of low-dose and low-dose-rate radiation are still rather controversial. Therefore, in this study we investigated the effects of low-dose-rate radiation on zebrafish neurodevelopment and the role of miRNAs in radiation-induced neurodevelopment. Zebrafish embryos received prolonged gamma-ray irradiation (0 mGy/h, 0.1 mGy/h, 0.2 mGy/h, 0.4 mGy/h) during development. Neurodevelopmental indicators included mortality, malformation rate, swimming speed, as well as the morphology changes of the lateral line system and brain tissue. Additionally, spatiotemporal expression of development-related miRNAs (dre-miR-196a-5p, dre-miR-210-3p, dre-miR-338) and miRNA processing enzymes genes (Dicer and Drosha) were assessed by qRT-PCR and whole mount in situ hybridization (WISH). The results revealed a decline in mortality, malformation and swimming speed, with normal histological and morphological appearance, in zebrafish that received 0.1 mGy/h; however, increased mortality, malformation and swimming speed were observed, with pathological changes, in zebrafish that received 0.2 mGy/h and 0.4 mGy/h. The expression of miRNA processing enzyme genes was altered after irradiation, and miRNAs expression was downregulated in the 0.1 mGy/h group, and upregulated in the 0.2 mGy/h and 0.4 mGy/h groups. Furthermore, ectopic expression of dre-miR-210-3p, Dicer and Drosha was also observed in the 0.4 mGy/h group. In conclusion, the effect of low-dose and low-dose-rate radiation on neurodevelopment follows the threshold model, under the regulation of miRNAs, excitatory effects occurred at a dose rate of 0.1 mGy/h and toxic effects occurred at a dose rate of 0.2 mGy/h and 0.4 mGy/h.

Characterization of paramyosin protein structure and gene expression during myogenesis in Pacific oyster (Crassostrea gigas).

Paramyosin is a key component of thick filaments in invertebrate muscles. In this study, we isolated the full length cDNA of paramyosin from Pacific oyster (Crassostrea gigas), and determined its pattern of expression during myogenesis. The full length paramyosin (CgPM) cDNA contains an open reading frame (ORF) of 2586bp encoding a 861-amino acid protein. Sequence analysis revealed an assembly competence domain (ACD) and a heptad repeat (d-e-f-g-a-b-c) with 28-residue repeat zones in the CgPM primary structure, a characteristic of coiled-coil protein. Quantitative analysis of CgPM expression revealed a sharp increase in trochophore stage, and peaked at the D-shaped stage. Strong CgPM expression was found in smooth adductor muscle, followed by striated adductor muscle and mantle tissue. Whole-mount in situ hybridization (WISH) showed a restricted pattern of CgPM expression in adductor muscle, larval velum retractor and foot muscles at the umbo and eyed larval stages. These data indicate that CgPM is strongly expressed during larval myogenesis in C. gigas, which provides the basis for further functional studies of paramyosin in oyster to better understand the molecular and cellular mechanisms of muscle formation in mollusks.

Dead-End (dnd) Gene Cloning and Gonad-Specific Expression Pattern in Starry Flounder (Platichthys stellatus).

Simple SummaryThe dead end (dnd) gene encodes an RNA-binding protein that plays a role for migration of primordial germ cells (PGCs) to the gonadal region during embryogenesis in vertebrates. Here, the starry flounder (Platichthys stellatus) dead end (psdnd) gene was characterized and its expression patterns were analyzed. Full-length psdnd mRNA was 1495 bp long, encoding 395 amino acids. psdnd was only expressed in gonadal tissues, we detected no psdnd expression in somatic cells. Furthermore, psdnd was strongly expressed during early embryogenesis. Our findings suggest that psdnd expression is gonad-specific and could therefore be used as a germ cell marker in starry flounder.dnd is a germline-specific maternal RNA expressed in various vertebrate classes, which encodes an RNA-binding protein that is essential for PGC migration. The purpose of this study is fundamental research about starry flounder dnd gene for germ cell marker development. In this study, we cloned and analyzed the expression levels of Platichthys stellatus dead end (psdnd) in various tissues and embryonic stages. The psdnd gene was isolated from starry flounder ovaries, cloned into a pGEM-t vector, and sequenced. Full-length of psdnd cDNA was 1495 bp long, encoding 395 amino acids. psdnd expression levels were investigated by real-time polymerase chain reaction (qRT-PCR) in various tissues and embryo developmental stages. psdnd transcripts were detected in the testes and ovaries, but not in somatic tissues. Embryonic psdnd expression levels were higher during early embryo development stages than during late embryogenesis; psdnd expression was highest at the 1 cell stage, then gradually decreased throughout the subsequent developmental stages. The spatial expression pattern was analyzed by whole-mount in situ hybridization (WISH). The psdnd transcripts migration pattern was similar with zebrafish (Danio rerio). Our results suggest that psdnd may function as a germ cell-specific marker.

Dhx15 regulates zebrafish definitive hematopoiesis through the unfolded protein response pathway

Gene alterations are recognized as important events in acute myeloid leukemia (AML) progression. Studies on hematopoiesis of altered genes contribute to a better understanding on their roles in AML progression. Our previous work reported a DEAH box helicase 15 (DHX15) R222G mutation in AML patients, and we showed DHX15 overexpression is associated with poor prognosis in AML patients. In this work, we further study the role of dhx15 in zebrafish developmental hematopoiesis by generating dhx15−/− zebrafish using transcription activator‐like effector nuclease technology. Whole‐mount in situ hybridization (WISH) analysis showed hematopoietic stem/progenitor cells were dramatically perturbed when dhx15 was deleted. Immunofluorescence staining indicated inhibited hematopoietic stem/progenitor cell (HSPC) proliferation instead of accelerated apoptosis were detected in dhx15−/− zebrafish. Furthermore, our data showed that HSPC defect is mediated through the unfolded protein response (UPR) pathway. DHX15 R222G mutation, a recurrent mutation identified in AML patients, displayed a compromised function in restoring HSPC failure in dhx15−/−; Tg (hsp: DHX15 R222G) zebrafish. Collectively, this work revealed a vital role of dhx15 in the maintenance of definitive hematopoiesis in zebrafish through the unfolded protein respone pathway. The study of DHX15 and DHX15 R222G mutation could hold clinical significance for evaluating prognosis of AML patients with aberrant DHX15 expression.

IL6 Signaling Regulates Embryonic Hematopoietic Stem Cell Emergence

Recent studies have illustrated the significance of some proinflammatory factors in regulating embryonic hemtopoietic stem cells (HSCs) production ,such as TNFα、IFNγ and TLR4.Il6 is a powerful proinflammatory cytokine that plays a pivotal role in the regulation of immunity,hematopoiesis and other physiological processes.Il6-deficient mice leads to impaired survival and self-renewal of HSCs and early progenitors.Whereas Il6 signaling regulates aspects of adult hematopoiesis,little is known about its role in the developmental specification of HSCs.This has led us to explore the impact of loss of Il6 signaling on embryonic HSC emergence by injecting specific antisense morpholinos (MOs) into zebrafish embryos at 1-2 cell stage.Here we report that MO knockdown of Il6r and Il6 strongly reduced the number of cmyb+HSCs in or near the floor of the dorsal aorta (DA) at 36 hr post fertilization (36hpf) by wholemount in situ hybridization (WISH)compared with their standard MO(Std MO)siblings(Fig.1a).Consistent with the results above,the number of double-positive cmyb+kdrl+ HSCs was reduced about 50% in the floor of the DA in cmyb:GFP;kdrl:mCherry double transgenic embryos at 48 hpf by confocal microscopy when compared to control embryos(Fig. 1b).To distinguish these reductions were due to a defect in the initial specification of HSCs or in their subsequent maintenance,we performed WISH for runx1, both Il6r- and Il6- deficient embryos showed significant reduction in the number of runx1+ HSCs in the aortic floor at 24 and 28hpf.Meanwhile,Il6r and Il6 morphants had sustained reductions in CD41:eGFP+ HSCs colonizing the caudal hematopoietic tissue (CHT) at 72 hpf using transgenic CD41:eGFP animals. Additionally ,expression of rag1 in thymus was completely or nearly absent in Il6r- and Il6-deficient embryos at 96 hpf(Fig. 1c), further surpporting that T-cell defects in these embryos resulted from early HSPC defects. Together, these results demonstrate that Il6 signaling pathway is important both for early specification and subsequent maintenance of HSC fate.To begin to determine the mechanism by which Il6 signaling regulates embryonic HSCs emergence in vivo,we first tested the impact of loss of Il6 signaling on vasculogenesis.No vascular abnormalities or impaired arterial specification were observed in Il6r- or Il6-deficient embryos at 28hpf when assayed by WISH for kdrl,efnb2a and dlc at the MO doses used in this study,suggesting that the function of Il6 signaling is required specifically during HSC development independently of their role in developing vasculature.Besides,we found that loss of Il6 signaling caused no increased apoptotic endothelial cells within the DA.Primitive hematopoiesis was also unaffected in our study.Lastly,to investigate the connection between Notch and Il6 signaling in HSC specification,we utilized tp1:eGFP;kdrl:mCherry double transgenic embryos and found that the double-positive tp1+kdrl+ Notch signaling-activated HSCs in the ventral floor of the DA had no significant change at 32-36hpf when blocking the IL6 signaling;embryos treated with notch signaling inhibitor DAPT developed fewer cmyb+ HSCs in the ventral floor of DA at 48hpf by WISH than DMSO-treated siblings;however,when the embryos were injected 200pg Il6r mRNA simutaneously, the HSC defect caused by DAPT could be partially rescued (Fig. 1d).In summary,our studies unravel an unexpected developmental role for Il6 signaling and place its action downstream of Notch signaling to control HSC emergence.Figure legendFigure 1. a)Representative cmyb WISH images and qualitative phenotype distribution (normal/down,n≥30 embryos/condition)of Il6r,Il6 and std MO-injected embryos at 36 hpf. b) Maximum projections of 48 hpf cmyb:GFP;kdrl:mCherry double-transgenic embryos injected with Std, Il6r, and Il6 MOs. Arrowheads denote cmyb+kdrl+ HSCs along the DA (left figure).Enumeration of cmyb+kdrl+ HSCs shown in(right figure).Error bars represent mean ± SEM of Std (n = 24), Il6r(n = 22), and Il6(n = 24) morphants. c)phenotype distribution(normal/down/absent,n≥30 embryos/condition) of rag1 expression in Il6r,Il6 and std MO-injected embryos at 96 hpf.d)WISH samples and phenotype distribution(high/medium/low,n≥30 embryos/condition)of cmyb expression in DMSO-treated,DAPT-treated,DAPT-treated and Il6r mRNA-injected simutaneously embryos at 48 hpf . [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Dysregulation of Follistatin Promotes Leukemogenesis in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is characterized by an abnormal increase of myeloblasts in bone marrow (BM) and peripheral blood (PB). Chemotherapy and allogeneic hematopoietic stem cell transplantation (HSCT) are the mainstays of treatment. However, these approaches have reached an impasse and despite an initial remission, leukemia relapses are frequent, with an overall cure rate of only 30-40% (Rowe and Tallman 2010). Internal tandem duplication (ITD) of the fms-like tyrosine kinase 3 (FLT3) gene is the commonest mutation in AML, occurring in nearly 30% cases with inferior disease-free and overall survivals (Boissel et al. 2002; Meshinchi et al. 2001). FLT3-ITD as a therapeutic target has been tested in phase I/II clinical trials (Man et al. 2012; Fischer et al. 2010; Levis et al. 2011; Cortes et al. 2011). In most circumstances, there was an initial response to FLT3 inhibitors with reduction or clearance of myeloblasts in BM and PB. However, resistance invariably developed despite continuous treatment (Kindler, Lipka, and Fischer 2010). Therefore, the identification of novel therapeutic targets for FLT3-ITD+ AML becomes urgent.In our previous study (He et al. 2014), we showed that over-expression of human leukemic gene FLT3-ITD resulted in significant myeloid cells expansion which could be effectively rescued by FLT3-ITD inhibitor AC220 treatment. Interestingly, a proportion of zebrafish embryos expressing FLT3-ITD showed double-headed and axis-duplication phenotype as shown by the whole mount in situ hybridization (WISH) of a notochord-specific marker col9a2 . Mechanistically, FLT3-ITD-induced axis-duplication was correlated with the expansion of Spemann's Organizer as shown by the ectopic expression of Spemann's Organizer-specific marker goosecoid (gsc) by WISH as well as by the -2,067gsc-GFP reporter assay. It has been reported that some key signaling pathways are conserved between embryogenesis and tumorigenesis. We hypothesized that certain Spemann's Organizers were dysregulated in FLT3-ITD+ AML.To test this hypothesis, we performed an in silico analysis to quantify the gene expression of Spemann's Organizers in normal HSC and AML by the public database BloodSpot. Intriguingly, the Activin antagonist Follistatin (FST) was significantly increased in hematological malignancies, comparing to the normal HSC. In human malignancies, FST has been shown to express in prostate (Sepporta et al. 2013; Tumminello et al. 2010), ovarian (Ren et al. 2012; Karve et al. 2012; Di Simone et al. 1996), and liver cancers (Patella et al. 2006; Rossmanith et al. 2002) promote cancer cell growth (Gao et al. 2010; Rossmanith et al. 2002). However, the role of FST in human AML and other hematological malignancies is currently unknown. Firstly, we confirmed the upregulation of FST in AML by real-time quantitative PCR, Western Blotting, and immunofluorescence imaging. Mechanistically, FLT3-ITD up-regulated FST via the phosphorylation of CREB as demonstrated by ChIP-PCR. Moreover, the upregulation of FST by FLT3-ITD was effectively blocked by the pharmacological inhibition of CREB in a dosage-dependent manner. FST gene knock-down (by shRNA) or knock-out (by CRISPR/Cas9) in FLT3-ITD+ MOLM-13 AML cell line significantly reduced leukemic growth in vitro, reduced leukemic burden, and prolonged survival of mice in the xenotransplantation experiment. Over-expression of FST in MOLM-13 and ML-2 AML cell lines promoted leukemic growth in vitro and in vivo xenograft mouse model via AKT/p70S6K/mTOR signaling. Furthermore, proteins (RPS19, RPS6, RPS13, RPL6, RPS21, RPSA, RPS18, RPLP2, RPL10A, WIBG, and PPP2R1A) associated with nonsense-mediated decay of mRNA were decreased after FST over-expression in ML-2 AML cells, while the chemoresistant and anti-apoptotic protein CD44 was consistently increased by a label-free proteomics analysis. Notably, the serum FST levels were significantly increased in a patient-derived xenograft AML mouse model. Consistently, plasma FST levels from FLT3-ITD+ AML patients were significantly reduced after an FLT3-ITD inhibitor quizartinib treatment in a phase I/II clinical trial.In conclusion, the CREB targeted gene FST promotes AML cell growth via AKT/p70S6K/mTOR signaling and CD44-mediated anti-apoptosis. Therefore, FST may be a promising therapeutic target for AML treatment. DisclosuresNo relevant conflicts of interest to declare.

Serotonin stimulates Echinococcus multilocularis larval development

BackgroundSerotonin is a phylogenetically ancient molecule that is widely distributed in most metazoans, including flatworms. In addition to its role as a neurotransmitter, serotonin acts as a morphogen and regulates developmental processes. Although several studies have focused on the serotonergic nervous system in parasitic flatworms, little is known on the role of serotonin in flatworm development.MethodsTo study the effects of serotonin on proliferation and development of the cestode Echinococcus multilocularis, we cloned the genes encoding the E. multilocularis serotonin transporter (SERT) and tryptophan hydroxylase (TPH), analyzed gene expression by transcriptome analysis and whole mount in situ hybridization (WMISH) and performed cell culture experiments.ResultsWe first characterized orthologues encoding the SERT and TPH, the rate-limiting enzyme in serotonin biosynthesis. WMISH and transcriptomic analyses indicated that the genes for both SERT and TPH are expressed in the parasite nervous system. Long-term treatment of parasite stem cell cultures with serotonin stimulated development towards the parasite metacestode stage. Mature metacestode vesicles treated with serotonin showed increased rates of incorporation of the thymidine analogue 5-ethynyl-2′-deoxyuridine (EdU), indicating stimulated cell proliferation. In contrast, treatment with the selective serotonin reuptake inhibitor paroxetine strongly affected the viability of parasite cells. Paroxetine also caused structural damage in metacestode vesicles, suggesting that serotonin transport is crucial for the integrity of parasite vesicles.ConclusionsOur results indicate that serotonin plays an important role in E. multilocularis development and proliferation, providing evidence that the E. multilocularis SERT and TPH are expressed in the nervous system of the protoscolex. Our results further suggest that the E. multilocularis SERT has a secondary role outside the nervous system that is essential for parasite integrity and survival. Since serotonin stimulated E. multilocularis metacestode development and proliferation, serotonin might also contribute to the formation and growth of the parasite in the liver.Graphical

Xenopus gpx3 Mediates Posterior Development by Regulating Cell Death during Embryogenesis.

Glutathione peroxidase 3 (GPx3) belongs to the glutathione peroxidase family of selenoproteins and is a key antioxidant enzyme in multicellular organisms against oxidative damage. Downregulation of GPx3 affects tumor progression and metastasis and is associated with liver and heart disease. However, the physiological significance of GPx3 in vertebrate embryonic development remains poorly understood. The current study aimed to investigate the functional roles of gpx3 during embryogenesis. To this end, we determined gpx3’s spatiotemporal expression using Xenopus laevis as a model organism. Using reverse transcription polymerase chain reaction (RT-PCR), we demonstrated the zygotic nature of this gene. Interestingly, the expression of gpx3 enhanced during the tailbud stage of development, and whole mount in situ hybridization (WISH) analysis revealed gpx3 localization in prospective tail region of developing embryo. gpx3 knockdown using antisense morpholino oligonucleotides (MOs) resulted in short post-anal tails, and these malformed tails were significantly rescued by glutathione peroxidase mimic ebselen. The gene expression analysis indicated that gpx3 knockdown significantly altered the expression of genes associated with Wnt, Notch, and bone morphogenetic protein (BMP) signaling pathways involved in tailbud development. Moreover, RNA sequencing identified that gpx3 plays a role in regulation of cell death in the developing embryo. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and phospho-histone 3 (PH3) staining confirmed the association of gpx3 knockdown with increased cell death and decreased cell proliferation in tail region of developing embryos, establishing the involvement of gpx3 in tailbud development by regulating the cell death. Furthermore, these findings are inter-related with increased reactive oxygen species (ROS) levels in gpx3 knockdown embryos, as measured by using a redox-sensitive fluorescent probe HyPer. Taken together, our results suggest that gpx3 plays a critical role in posterior embryonic development by regulating cell death and proliferation during vertebrate embryogenesis.

Schistosoma mansoni venom allergen-like protein 6 (SmVAL6) maintains tegumental barrier function

The Schistosoma mansoni venom allergen-like protein (SmVAL) superfamily is a collection of at least 29 molecules that have been classified into two distinctive groups (Group 1 and Group 2 SmVALs). The fundamental basis for SmVAL segregation relates to signal peptide and conserved cysteine retention (present in all Group 1 SmVALs, but absent in all Group 2 SmVALs). These structural differences have led to the hypothesis that most Group 1 SmVALs, found as components of schistosome excretory/secretory (E/S) products, predominantly interact with their environment (intermediate or definitive hosts) whereas the Group 2 SmVALs are retained within the schistosome to fulfil parasite-related functions. While experimental evidence to support Group 1 SmVAL/host interactions is growing, similar support for identification of parasite-related Group 2 SmVAL functions is currently lacking. By applying a combination of approaches to the study of SmVAL6, we provide the first known evidence for an essential function of a Group 2 SmVAL in schistosome biology. After whole mount in situ hybridisation (WISH) localised Smval6 to the anterior region of the oesophageal gland (AOG) and cells scattered through the mesenchyme in adult schistosomes, short interfering RNA (siRNA)-mediated silencing of Smval6 was employed to assess loss of function phenotypes. Here, siSmval6-mediated knockdown of transcript and protein levels led to an increase in tegumental permeability as assessed by the quantification of TAMRA-labelled dextran throughout sub-tegumental cells/tissues. Yeast two hybrid screening using SmVAL6 as a bait revealed Sm14 (a fatty acid binding protein) and a dynein light chain (DLC) as directly interacting partners. Interrogation of single-cell RNA-seq (scRNA-seq) data supported these protein interactions by demonstrating the spatial co-expression of Smval6/dlc/Sm14 in a small proportion of adult cell types (e.g. neurons, tegumental cells and neoblasts). In silico modelling of SmVAL6 with Sm14 and DLC provided evidence that opposing faces of SmVAL6 were likely responsible for these protein/protein interactions. Our results suggest that SmVAL6 participates in oesophageal biology, formation of higher order protein complexes and maintenance of tegumental barrier function. Further studies of other Group 2 SmVALs may reveal additional functions of this enigmatic superfamily.

Zrsr2 Deficient Zebrafish Display Hematopoietic Defects and U12-Type Intron Retention in mRNA Processing Genes

ZRSR2 is a small nuclear riboprotein necessary for assembly of the minor spliceosome and subsequent splicing of U12-type introns. It is involved in the recognition of 3' splice sites during the assembly of the spliceosome. Somatic mutations in ZRSR2 gene are present in hematopoietic malignancies, most notably myelodysplastic syndrome (MDS). These mutations are spread throughout the gene, indicating that its function is critical for hematopoiesis. In spite of these clinical associations, the role of ZRSR2 in hematopoiesis has not been well studied. Zebrafish make an excellent animal model for investigating this gene as all of the functional domains in the human protein are conserved with a high level of protein similarity in zebrafish. Additionally, hematopoietic development is well understood in this species. We used CRISPR/Cas9 to generate a zebrafish zrsr2 knockout model with an 11 base pair deletion (zrsr2Δ11) that results in frameshift with premature truncation and loss of all functional domains (p.W167fs*175). The zrsr2Δ11/Δ11 mutants began to appear morphologically different from their siblings by 4 days post fertilization (dpf), showing aberrant development in the mandible and pharyngeal arches. The zrsr2Δ11/Δ11 mutants developed mild to severe edema by 6 dpf and died by 8 dpf. To understand its role in hematopoiesis, we performed o-dianisidine staining and whole mount in situ hybridization (WISH) with lineage-specific markers during embryonic development. Our data showed that zrsr2Δ11/Δ11 embryos exhibit mild anemia by 2 dpf, suggesting that primitive hematopoiesis is defective. WISH showed that zrsr2Δ11/Δ11 mutants have normal early hematopoietic stem cell (HSC) emergence (c-myb) at 36 hours post fertilization. However, these cells are absent through 3 and 5 dpf. At 5 dpf zrsr2Δ11/Δ11 mutants had severely reduced expression of hematopoietic markers for erythroid (hbae1), lymphoid (rag1), and myeloid (mpo) lineages. This verifies that knockout of zrsr2 in zebrafish disrupted normal hematopoiesis and leads to cytopenias - emulating the symptoms of MDS. We then investigated the transcriptional and splicing changes underlying these phenotypes using RNA sequencing on zrsr2Δ11/Δ11 mutant embryos at 3dpf. We found a total of 285 intron retention events in zrsr2Δ11/Δ11 mutants as compared to the control embryos, with about 76% of those events occurring within U12-type introns. Genes with retained introns were associated with mRNA decay and destabilization. We also found that genes related to cell cycle arrest were upregulated in zrsr2Δ11/Δ11 mutants . These results indicate that aberrant mRNA splicing and cell cycle deregulation contribute to arrested hematopoiesis when zrsr2 is knocked out in zebrafish. Disclosures No relevant conflicts of interest to declare.

Gdnf affects early diencephalic dopaminergic neuron development through regulation of differentiation-associated transcription factors in zebrafish.

Glial cell line-derived neurotrophic factor (GDNF) has been reported to enhance dopaminergic neuron survival and differentiation in vitro and in vivo, although those results are still being debated. Glial cell line-derived neurotrophic factor (gdnf) is highly conserved in zebrafish and plays a role in enteric nervous system function. However, little is known about gdnf function in the teleost brain. Here, we employed clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 to impede gdnf function in the maintenance of dopaminergic neuron development. Genotyping of gdnf crispants revealed successful deletions of the coding region with various mutant band sizes and down-regulation of gdnf transcripts at 1, 3 and 7day(s) post fertilization. Notably, ~20% reduction in ventral diencephalic dopaminergic neuron numbers in clusters 8 and 13 was observed in the gdnf-deficient crispants. In addition, gdnf depletion caused a modest reduction in dopaminergic neurogenesis as determined by 5-ethynyl-2'-deoxyuridine pulse chase assay. These deleterious effects could be partly attributed to deregulation of dopaminergic neuron fate specification-related transcription factors (otp,lmx1b,shha,and ngn1) in both crispants and established homozygous mutants with whole mount in-situ hybridization (WISH) on gdnf mutants showing reduced otpb and lmx1b.1 expression in the ventral diencephalon. Interestingly, locomotor function of crispants was only impacted at 7dpf, but not earlier. Lastly, as expected, gdnf deficiency heightened crispants vulnerability to 1-methyl-4-phenylpyridinium toxic insult. Our results suggest conservation of teleost gdnf brain function with mammals and revealed the interactions between gdnf and transcription factors in dopaminergic neuron differentiation.

Expression dynamics of genes in the hypothalamic-pituitary-thyroid (HPT) cascade and their responses to 3,3′,5-triiodo-l-thyronine (T3) highlights potential vulnerability to thyroid-disrupting chemicals in zebrafish (Danio rerio) embryo-larvae

Some chemicals in the environment disrupt thyroid hormone (TH) systems leading to alterations in organism development, but their effect mechanisms are poorly understood. In fish, this has been limited by a lack of fundamental knowledge on thyroid gene ontogeny and tissue expression in early life stages. Here we established detailed expression profiles for a suite of genes in the hypothalamic-pituitary-thyroid (HPT) axis of zebrafish (Danio rerio) between 24–120 h post fertilisation (hpf) and quantified their responses following exposure to 3,3’,5-triiodo-L-thyronine (T3) using whole mount in situ hybridisation (WISH) and qRT-PCR (using whole-body extracts). All of the selected genes in the HPT axis demonstrated dynamic transcript expression profiles across the developmental stages examined. The expression of thyroid receptor alpha (thraa) was observed in the brain, gastrointestinal tract, craniofacial tissues and pectoral fins, while thyroid receptor beta (thrb) expression occurred in the brain, otic vesicles, liver and lower jaw. The TH deiodinases (dio1, dio2 and dio3b) were expressed in the liver, pronephric ducts and brain and the patterns differed depending on life stage. Both dio1 and dio2 were also expressed in the intestinal bulb (96–120 hpf), and dio2 expression occurred also in the pituitary (48–120 hpf). Exposure of zebrafish embryo-larvae to T3 (30 and 100 μg L−1) for periods of 48, 96 or 120 hpf resulted in the up-regulation of thraa, thrb, dio3b, thyroid follicle synthesis proteins (pax8) and corticotropin-releasing hormone (crhb) and down-regulation of dio1, dio2, glucuronidation enzymes (ugt1ab) and thyroid stimulating hormone (tshb) (assessed via qRT-PCR) and responses differed across life stage and tissues. T3 induced thraa expression in the pineal gland, pectoral fins, brain, somites, gastrointestinal tract, craniofacial tissues, liver and pronephric ducts. T3 enhanced thrb expression in the brain, jaw cartilage and intestine, while thrb expression was suppressed in the liver. T3 exposure suppressed the transcript levels of dio1 and dio2 in the liver, brain, gastrointestinal tract and craniofacial tissues, while dio2 signalling was also suppressed in the pituitary gland. Dio3b expression was induced by T3 exposure in the jaw cartilage, pectoral fins and brain. The involvement of THs in the development of numerous body tissues and the responsiveness of these tissues to T3 in zebrafish highlights their potential vulnerability to exposure to environmental thyroid-disrupting chemicals.

Hatching gland development and hatching in zebrafish embryos: A role for zinc and its transporters Zip10 and Znt1a

Zinc transporters of the ZIP (Slc39, importers) and ZnT (Slc30, exporters) protein families have evolutionary conserved roles in biology. The aim of the present study was to explore the role of zinc, and zinc transporters Zip10 and Znt1a in zebrafish hatching gland development and larval hatching. In the study, knockdown of genes for Zip10 and Znt1a in zebrafish embryos was achieved using morpholino-modified oligonucleotides. A partial loss-of-function Znt1a mutant (Znt1asa17) allowed comparison with the Znt1a morphant. Free Zn2+ in embryos and apoptosis were investigated using fluorescent dyes whereas gene expression was investigated by whole-mount in situ hybridization (WISH). The results showed high levels of free Zn2+ in the hatching gland cells (HGC) along with abundant expression of zip10 and znt1a in normal embryo. Knockdown of zip10 reduced free Zn2+ in HGC, ceased their normal developmental apoptosis, and resulted in displacement and later disappearance of hatching glands and hatching enzymes he1a and catL1b, and inability to hatch. Conversely, knockdown of znt1a or the Znt1asa17 mutation accelerated hatching and coincided with high expression of hatching enzymes and free Zn2+ in the HGC. Thus, Zip10 and free Zn2+ in the HGC are required both for their development and function. This study also demonstrated the opposite functions of the two zinc transporters, ZIP10 and ZnT1 as well as shedding light on the role of Zn2+ in regulation of the human hatching enzyme homologue, ovastacin, which is activated by zinc and cleaves the zona pellucida protein, ZP2, to prevent polyspermy.

Vitamin E is Necessary to Protect Neural Crest Cells in Developing Zebrafish Embryos

Abstract Objectives Vitamin E (VitE) deficiency causes vertebrate embryonic lethality. The alpha-tocopherol transfer protein (Ttpa) likely regulates VitE distribution in the early zebrafish embryo because Ttpa knockdown causes impaired nervous system development and embryonic death by 15–18 hours post-fertilization (hpf). We propose that VitE is necessary for normal brain and peripheral nervous system development. Methods Zebrafish embryos are obtained from adults fed either VitE sufficient (E+) or deficient (E–) diets for at least 80 days. Embryos at 12 and 24 hpf are subjected to RNA whole mount in situ hybridization (WISH). RNA is also collected from embryos at 12, 18 and 24 hpf for RT-qPCR of specific targets. Results At 12 hpf, the midbrain-hindbrain boundary and otic placodes are malformed in E– embryos, as shown by Pax2a expression. Similarly, Sox10 expression shows that E– embryos lack clear neural plate borders. Nonetheless, in 12 hpf E + and E− embryos Ttpa is localized similarly throughout the nervous system. Pax2a expression initiates collagen formation in the developing notochord. Collagen genes, col2a1a and col9a2, expression patterns showed abnormal notochord structures in 24 hpf E– embryos. At 24 hpf in E + embryos, Sox10 expressing-neural crest cells are localized both in the central nervous system and dorsal root ganglia (DRG), while the Sox10 signal is diminished in E– embryos in both the DRG and early enteric nervous system. At 24 hpf, Ttpa expression outlines the brain ventricle borders; critically E– embryos show reduced Ttpa signal and impaired ventricle closing. Gene expression by qPCR will be used to confirm these results. Conclusions This VitE deficient embryo model suggests that the carefully programmed development of the nervous system is distorted due to lack of adequate VitE. Thus, Ttpa and VitE are critical molecules for neural plate and neural tube formation, and neural crest cell migration. Funding Sources The authors received no specific funding for this work.

Whole-Mount In Situ Hybridization in Post-Implantation Staged Mouse Embryos.

Understanding RNA expression in space and time is a key initial step in dissecting gene function. The ability to visualize gene expression in whole-tissue or whole-specimen preparations, called in situ hybridization (ISH), was first developed 50 years ago. Two decades later, these protocols were adapted to establish robust methods for whole-mount ISH to murine embryos. The precise protocols vary somewhat between early-gestation and mid-gestation mouse embryos; the protocol presented here is optimal for use with post-implantation stage mouse embryos (stages 5.5-9.5 dpc). Routine uses of whole-mount ISH include documenting the wild-type expression pattern of individual genes and comparison of the expression pattern of signature genes (i.e., those that identify particular cells and tissues within an embryo) between wild-type and mutant embryos as part of a phenotyping experiment. This technique remains a mainstay of developmental biology studies and complements the massively parallel assessment of gene expression from dissociated tissues and cells via RNA-sequencing techniques. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Dissection of post-implantation (5.5-9.5 dpc) murine embryos Basic Protocol 2: Whole-mount in situ hybridization in post-implantation embryos Basic Protocol 3: Visualization of post-WMISH embryos Support Protocol 1: Creation of siliconized glass pipettes Support Protocol 2: Creation of embryo powder.

Identification and functional study of the Schistosoma japonicum epidermal growth factor receptor gene

To characterize the epidermal growth factor receptor (EGFR) gene in Schistosoma japonicum (SjEGFR gene) and investigate the role of the EGFR gene in regulating the growth, reproductive system, maturation and fecundity of S. japonicum. Rapid amplification of cDNA ends (RACE) was performed to obtain the full length of the SjEGFR gene, and the SjEGFR gene expression was quantified in different developmental stages of S. japonicum using a quantitative real-time PCR (qPCR) assay. The tissue localization of the SjEGFR gene was detected in 22-day parasite using whole-mount in situ hybridization (WISH). Following RNA interference (RNAi)-induced knockdown of the SjEGFR gene, the worm length, pairing rate and worm burden of S. japonicum were measured, and the worm morphology was observed using optical microscopy and confocal microscopy. The SjEGFR gene was identified with a conserved tyrosine-kinase active site, and the SjEGFR gene expression was detected at various developmental stages in male and female parasites. WISH showed that the transcript of the SjEGFR gene was localized on the tegument and in the digestive organs of S. japonicum. RNAi-induced SjEGFR knockdown resulted in marked suppression of the worm growth, smaller size of male testicles that contained more immature spermatocytes, and apparent impairment of ovary and vitelline gland development. In addition, no eggs were found in the uterus of SjEGFR knocked-down female parasites, indicating the interruption of egg production. Inhibition of SjEGFR expression may remarkably suppress the growth and maturation of S. japonicum, and interrupt the egg production.

Zebrafish ambra1a and ambra1b Silencing Affect Heart Development.

In zebrafish, two paralogous genes, activating molecule in beclin-1 (BECN1)-regulated autophagy ambra1a and ambra1b, both required for the autophagic process and during development, encode the protein AMBRA1, a positive regulator of early steps of autophagosome formation. As transcripts for both genes are expressed during embryogenesis in the heart region, in this work, we investigated the effects of ambra1a and ambra1b knockdown on heart development by means of morpholino oligonucleotides (MOs). Silencing of the two proteins by MOs directed against the ATG translation initiation codon affects cardiac morphogenesis, resulting in a small, string-like heart with pericardial edema, whereas treatment with splice-blocking MOs does not lead to overt cardiac phenotypes, thus revealing the relevance of maternally supplied ambra1 transcripts for heart development. Co-injection of both ATG-MOs determines a more severe cardiac phenotype, with prominent pericardial edema. Whole-mount in situ hybridization (WMISH) for myosin light chain 7 (myl7), as well as ambra1 ATG-MO microinjection in zebrafish transgenic line expressing green fluorescent protein in the heart, revealed defects with the heart jogging process followed by imperfect cardiac looping. Moreover, WMISH of homeodomain transcription factor 2 isoform c (pitx2c) transcripts showed both bilateral and reversed pitx2c expression in morphants. The morphants' cardiac phenotypes were effectively rescued by co-injection of MOs with human AMBRA1 (hAMBRA1) messenger RNA (mRNA), pointing at the conservation of Ambra1 functions during evolution. Co-injections of ambra1 ATG-MOs with a hAMBRA1 mRNA mutated in the protein phosphatase 2a (PP2A) binding sites (hAMBRA1PXP) were not able to rescue the cardiac phenotypes, at the difference from wild-type hAMBRA1 mRNA, and treatment of zebrafish embryos with the specific PP2A inhibitor cantharidin resulted in similar developmental cardiac defects. These results suggest a critical role for AMBRA1 in vertebrate heart development, likely involving the binding site for the PP2A phosphatase.

The Pattern of EGR1 Expression During Chick Limb Development Suggests a Role in Regulating SHH Expression and/or Termination

Limb development is governed by a complex interplay between three signaling centers that mediate growth along three coordinate axes. The apical ectodermal ridge (AER), zone of polarizing activity (ZPA) and the dorsal ectoderm regulate the proximal‐distal, anterior‐posterior and dorsal‐ventral axes, respectively. Development occurs along these three axes to create an asymmetrical limb and is highly dependent on crosstalk between fibroblast growth factors (FGFs) from the AER and sonic hedgehog (SHH) from the ZPA. The mechanisms and intermediate players between FGF and SHH are poorly characterized. Previous work in our lab identified a number of FGF‐responsive candidate genes. Most notably early growth response 1 (EGR1), encoding a zinc finger type protein, was identified as the gene with the most robust induction in response to FGF. EGR1 has been shown to play a role in tendon development but to date its expression in the developing limb has not been well characterized. We found EGR1 to have low endogenous expression in the chick limb by whole mount in situ hybridization (WMISH). We highlighted the WMISH pattern of EGR1 using a 3 hr cycloheximide pretreatment to enhance RNA accumulation. This approach revealed that EGR1 is expressed subjacent to the AER and overlaps the ZPA during robust SHH expression (Hamburger‐Hamilton stage 17 (HH17) through HH24). In contrast, as SHH expression wanes (HH25–26), EGR1 expression recedes from the posterior ZPA, and persists in the anterior progress zone underlying the AER. In addition, EGR1 expression extends proximally from the anterior border of the progress zone. The reduction of EGR1 within the ZPA coincident with the decline in SHH may indicate a role for EGR1 in optimizing SHH expression and triggering termination.

Estrogen modulation of fate choice during kidney development

Vertebrate kidney formation is a nuanced process that directs renal progenitors to form the functional units of the kidney, known as nephrons, which are comprised of specialized cell types organized into discrete functional segments. The zebrafish embryo forms two nephrons by 24 hours post fertilization (hpf) and offers a simple but tractable genetic model to study nephron patterning. A chemical screen carried out by our lab coupled with whole mount in situ hybridization (WISH) identified 17ß‐estradiol (E2), the dominant form of estrogen in vertebrates, as a potential regulator of segmentation. Further investigation has revealed that E2 results in an expansion of the distal early (DE) and decrease of the distal late (DL) nephron segments due to alterations in cell number that were quantified with fluorescent ISH. Furthermore, E2 treated animals had expanded expression of the essential DE lineage transcription factor irx3b and its target, irx1a, suggesting E2 affects specification of DE precursors. To delineate the possible mechanism of action of E2’s renal effects, we conducted a targeted chemical screen with selective estrogen receptor modulators (SERMs) that exhibit preferential binding for known E2 receptors. The esr2 antagonist PHTPP similarly reduced the DE segment, suggesting that Esr2 may be involved in nephrogenesis. Consistent with this hypothesis, esr2a and esr2b transcripts are expressed during renal progenitor specification. To investigate which of these receptors (or both) are a major player in distal cell fate choice, we have created CRISPR/Cas9 mutants using guides designed for esr2a and esr2b, respectively. With additional genetic studies, we hope to further our understanding of the role of estrogen signaling in kidney development. Combined, these preliminary findings have relevance for understanding kidney disease models, as well as efforts to recapitulate development in vivo for drug discovery and regenerative therapies.Support or Funding InformationR01DK100237